The goal of this Phase I SBIR project is to develop a broadly applicable process for purpose of creating improved type 1.1 and 1.2 lipases used in pharmaceuticals and fine chemicals production. Lipases are useful for chemical synthesis because of their ability to resolve racemic mixtures of important chemical intermediates. Their use is limited because of their high cost. Much of cost associated with these enzymes is attributed to production and purification difficulties caused by the unique expression and folding requirements of these enzymes. The presence of unusual folding catalysts, poorly expressed in heterologous hosts, is required for these lipases to attain active conformations in the periplasm. The most efficient of current lipase production protocols require production of these lipase specific chaperones in separate fermentations, which are then used to refold the denatured lipase in vitro, adding a considerable cost. We will DNA sequences of the auto-folding mutants will be determined, enabling similar mutations to be recreated in other commercially important lipases. Additionally, we will create gene fusions that incorporate affinity tags to improve lipase purification, and allow the production of both the lipase and chaperone as a single polypeptide. PROPOSED COMMERCIAL APPLICATION: The novel enzymes and processes developed during the performance of this project will find immediate applications as biocatalysts for the production of enantio-pure chemical intermediates, and other industrial processes.